Medulloblastoma (MB) is a highly aggressive, malignant brain tumor in children with poor prognosis. Cyclin-dependent kinase 9 (CDK9), a serine-threonine kinase, is widely implicated in the control of basal gene expression by phosphorylating Serine 2 (Ser2) of the heptad repeat in the RNA Polymerase II (RNA Pol II) C-terminal domain (CTD). Although CDK9 plays a pathogenic role in various cancers, its function in MB remains unknown. Here, we show that CDK9 is highly expressed in MB tumors and increased CDK9 expression is correlated with high risk MB patients. CDK9 expression along with phospho-Ser2 RNA Pol II (pRNA Pol II ser2) and bromodomain-binding protein 4 (BRD4), which recruits CDK9, were elevated in multiple MB cell lines and in MB tumors originated spontaneously from Ptch1+/−p53−/− mice. Inhibition of CDK9 with LDC067 suppressed MB cell growth, reduced pRNA Pol II ser2 level and expression of oncogenic markers, including MYC. Moreover, LDC067 treatment synergistically sensitizes MB cells to chemotherapeutic agent cisplatin. Further, LDC067 in combination with BRD4 inhibitor decreased MB cells growth, delayed cell migration and attenuated pRNA Pol II ser2 occupancy to CCND1 and BCL2 gene promoters as revealed by chromatin immunoprecipitation assay (ChIP). Together, these findings highlight the importance of CDK9 in MB pathogenesis and suggest that it may serve as a promising therapeutic target for the treatment of MB. •CDK9 is overexpressed in MB tumors and is associated with high risk patients.•CDK9 inhibitor, LDC067, decreases serine2 phosphorylation of RNA pol II and reduces expression of MYC oncogene.•CDK9 inhibition suppresses MB cell growth and synergistically increases sensitivity of MB cells to cisplatin.•Combination of LDC067 with BRD4 inhibitor further impairs cell viability, migration and pRNA Pol II ser2 promoter binding.•CDK9 may serve as a novel therapeutic target for the treatment of MB.